System for delivering nucleic acids for suppressing target gene expression by utilizing endogenous chylomicron

ABSTRACT

The object of present invention is to provide a system that can deliver in vivo nucleic acids such as an siRNA for suppressing a target gene expression in vivo more safely and efficiently, and to provide an expression-suppressing agent and a pharmaceutical composition utilizing the system. An introduction substance into chylomiclon, particularly nucleic acids to which an alpha-tocopherol is bound for suppressing a target gene expression, can be delivered more safely and efficiently into hepatic cells in vivo by administering the nucleic aids under the condition where the production of chylomicron is induced in the body. Alternatively, alpha-tocopherol-bound nucleic acids are mixed with extracted chylomiclon, and then they are administered. Consequently, a target gene expression is suppressed, thereby a disease caused by an elevated expression of the target gene can be treated more safely and efficiently.

This application is a Continuation-in-Part of International ApplicationNo. PCT/JP2008/003523 filed Nov. 28, 2008 which claims benefit ofpriority to U.S. Provisional Application Ser. No. 60/990,796 filed Nov.28, 2007, the entire contents of which are hereby incorporated byreference herein.

TECHNICAL FIELD

The present invention relates to a system for delivering in vivo nucleicacids such as an siRNA for suppressing a target gene expression, and toan expression-suppressing agent and a pharmaceutical compositionutilizing the system.

BACKGROUND ART

An siRNA capable of suppressing the expression of a specific gene iswidely used as a research tool. An siRNA is also receiving attention forapplications to therapeutic agents for a wide variety of diseasesincluding tumors, infectious diseases, and hereditary diseases. The mostcritical problem in the clinical applications of an siRNA lies in thefact that an siRNA should be delivered specifically and efficiently to atarget cell in vivo. For example, a delivery method is known in which ansiRNA is delivered in vivo by means of a high-pressure and high-volumeintravenous injection of a synthetic siRNA, utilizing a viral vector.This method, however, uses a viral vector, and a restriction is imposedon the clinical applications of such method from the viewpoint of safetyand the like. Consequently, various non-viral systems have beendeveloped that can deliver an siRNA in vivo to the liver, tumor, orother tissues.

Examples of recently developed non-viral delivery systems include theones using: a cholesterol-siRNA complex (non-patent document 1); stablenucleic acid lipid particles (SNALP) (non-patent document 2);interfering nanoparticles (iNOP) (non-patent document 3) and the like.Among these non-viral delivery systems, SNALP has brought about a greatimprovement in that the use of SNALP for injecting a clinicallyappropriate amount of siRNAs has enabled the knockdown of a target mRNAin the liver. However, a therapeutic amount (2.5 mg/kg) of SNALP causeda significant damage to the liver in a crab-eating monkey, whosetransaminase level (ALT and AST) exceeded 1000 U/L 48 hours after theadministration. Further, a serious disadvantage of SNALP and iNOP isthat these delivery systems can only passively transfer an siRNA complexto the liver by utilizing lipophilic nature of the particles that couldcontribute to the toxicity.

Recently, new types of non-viral delivery systems have been reported,including an siRNA vector (RVG-9R) that can transfer an siRNA via areceptor (non-patent document 4) and “Dynamic PolyConjugate™” (Mirus)(non-patent document 5). The above-mentioned RVG-9R is a short peptidederived from a glycoprotein of rabies virus, added with 9 arginineresidues. By utilizing this RVG-9R, an siRNA can be transferred to anerve cell via an acetylcholine receptor. Meanwhile, the DynamicPolyConjugate contains a membrane-active form of polymer to which anN-acetylgalactosamine (NAG) is bound, as a ligand targeting a hepaticcell. While the use of these receptor-mediated delivery systems such asRVG-9R and Dynamic PolyConjugate can improve efficiency and specificityof an in vivo siRNA delivery to a target cell, artificially synthesizedvector molecules used in these systems still possess hazardous naturethat could cause serious side-effects particularly when the dose isincreased.

Most recently, a delivery method has been reported, which comprisesextracting endogenous lipoprotein, allowing LDL and HDL ex vivo to takein an siRNA to which a cholesterol molecule in the lipoprotein is bound,and introducing the siRNA to the liver via a lipoprotein receptor(non-patent document 6). This complex is taken in by the liver 5 to 8times more effectively as compared to a free cholesterol-siRNA. However,the complex is effective only to the extent that it can suppress thetarget gene in the liver by about 55% with a 13 mg/kg intravenous siRNAadministration, which is far from sufficient.

Under these circumstances, there have been demands for an siRNA deliverysystem that can efficiently and specifically deliver an siRNA in vivoand that has a lower risk of causing side-effects.

[Non-Patent Document 1]

-   Nature 432:173-178, 2004    [Non-Patent Document 2]-   Nature 441:111-114, 2006    [Non-Patent Document 3]-   ACS Chem. Biol. 2:237-241, 2007    [Non-Patent Document 4]-   Nature 448:39-43, 2007    [Non-Patent Document 5]-   Proc Natl Acad Sci USA. 104:12982-12987, 2007    [Non-Patent Document 6]-   Nature Biotechnology 25:1149-1157, 2007

DISCLOSURE OF THE INVENTION Technical Problem

The object of the present invention is to provide a system that can invivo deliver nucleic acids such as an siRNA for suppressing a targetgene expression in vivo more safely and efficiently, and to provide anexpression-suppressing agent and a pharmaceutical composition utilizingthe system.

Technical Solution

The present inventors administered a vitamin E-bound siRNA under thecondition where the production of endogenous chylomicron is induced invivo, and successfully delivered the siRNA in vivo more safely andefficiently. Consequently, the present inventors discovered that atarget-gene expression can be suppressed very efficiently, and thuscompleted the present invention.

More specifically, the present invention relates to an agent forsuppressing a target gene expression, comprising nucleic acids forsuppressing the target gene expression, wherein an introductionsubstance into chylomicron or chylomicron remnant is bound to thenucleic acids, and wherein the agent is administered to a vertebrateunder a condition in which a production of endogenous chylomicron isinduced in the vertebrate (1); the agent according to (1), wherein thecondition is a condition of within 12 hours after a lipid isadministered to the vertebrate (2); the agent according to (2), whereinthe lipid is administered in a form of an oral intake (3); the agentaccording to (1), wherein LPL inhibitor is administered to a vertebratebefore a production of endogenous chylomicron is induced in thevertebrate (4); The agent according to (1), wherein LPL and/or heparinis administered to a vertebrate before nucleic acids are administered tothe vertebrate. (5); the agent according to (1), wherein the vertebrateis allowed to be in a state of starvation prior to a lipidadministration. (6); the agent according to (1), wherein the nucleicacids to which an introduction substance into chylomicron is bound areadministered to a vertebrate, with the nucleic acids being mixed withconcentrated chylomicron obtained from a vertebrate (7); the agentaccording to (1), wherein the substance is a lipophilic vitamin orcholesterol (8); the agent according to (8), wherein the lipophilicvitamin is vitamin E (9); the agent according to (1), wherein thenucleic acids are one or more kinds of nucleic acids selected from thegroup consisting of siRNA, shRNA, antisense oligonucleotide, antagomir,nucleic-acid aptamer, ribozyme, and decoy (10); the agent according to(10), wherein the nucleic acids are siRNA (11); the agent according to(1), wherein the nucleic acids are RNA subjected to an anti-RNasetreatment (12); and the agent according to (12), wherein the anti-RNasetreatment is 2′-O-methylation treatment and/or thiophosphorylationtreatment (13).

The present invention also relates to a pharmaceutical compositioncomprising the agent according to (1) as an active ingredient (14).

The present invention further relates to a method for delivering in vivonucleic acids for suppressing a target gene expression, comprisingadministering the nucleic acids to a vertebrate under a condition inwhich a production of endogenous chylomicron or chylomicron remnant isinduced in the vertebrate, wherein an introduction substance intochylomicron is bound to the nucleic acids (15).

The present invention still further relates to a method for treating adisease that is ameliorated by suppressing a target gene expression,comprising administering nucleic acids for suppressing the target geneexpression to a vertebrate under a condition in which a production ofendogenous chylomicron or chylomicron remnant is induced in thevertebrate, wherein an introduction substance into chylomicron is boundto the nucleic acids (16); the method according to (15) or (16), whereinthe condition is a condition of within 12 hours after a lipid isadministered to a vertebrate (17); the method according to (17), whereinthe lipid is administered in a form of an oral intake (18); the methodaccording to (15) or (16), comprising administering LPL inhibitor to avertebrate before a production of endogenous chylomicron is induced inthe vertebrate. (19); The method according to (15) or (16), comprisingadministering LPL and/or heparin to a vertebrate before nucleic acidsare administered to the vertebrate. (20); the method according to (15)or (16), comprising allowing the vertebrate to be in a state ofstarvation prior to a lipid administration (21); the method according to(15) or (16), comprising administering to a vertebrate the nucleic acidsto which an introduction substance into chylomicron is bound, with thenucleic acids being mixed with concentrated chylomicron obtained from avertebrate (22); The method according to (22), comprising administeringLPL and/or heparin to a vertebrate before nucleic acids are administeredto the vertebrate. (23); the method according to (15) or (16), whereinthe substance is a lipophilic vitamin or cholesterol (24); the methodaccording to (24), wherein the lipophilic vitamin is vitamin E (25); themethod according to (15) or (16), wherein the nucleic acids are one ormore kinds of nucleic acids selected from the group consisting of siRNA,shRNA, antisense oligonucleotide, antagomir, nucleic-acid aptamer,ribozyme, and decoy (26); the method according to (26), wherein thenucleic acids are siRNA (27); and the method according to (15) or (16),wherein the nucleic acids are RNA subjected to an anti-RNase treatment(28).

BRIEF DESCRIPTION OF DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 This figure shows a general outline of the in vivo delivery of analpha-tocopherol (vitamin E)-bound siRNA (Toc-siRNA).

FIG. 2 This figure shows the transportation of an alpha-tocopherolbetween different types of tissues.

FIG. 3 This figure shows the stability and the expression-suppressingactivity of an siRNA chemically modified by a thiophosphorylation of theskeletal bond and 2′-O-methylation of the ribose of the nucleotides.

A: This figure shows the Toc-siRNA sequence targeted to the mouse apoBmRNA and the chemical modification of the sequence.

B. This figure shows the stability of the modified siRNA in a serum.

C: This figure shows the in vitro expression-suppressing efficiency ofthe modified siRNA.

FIG. 4 This figure shows the chemical structure of an alpha-tocopherol(vitamin E)-bound siRNA.

FIG. 5 This figure shows the interaction of an siRNA with a lipoprotein.

FIG. 6 This figure shows the interaction of a Toc-siRNA or Cho-siRNA anda lipoprotein (the results of an acrylamide gel electrophoresis).

FIG. 7 This figure shows the interaction of a Toc-siRNA or Cho-siRNA anda lipoprotein (the results of a fluorescence correlation spectroscopy).

FIG. 8 This figure shows the results of measurement of the diameter ofchylomicron by dynamic light scattering (DLS).

FIG. 9 This figure shows that the Toc-siRNA intravenously injected tothe mouse has been taken into the liver.

A: This figure shows the result of a fluorescence microscopicobservation of a liver-tissue segment after a Cy3-labeled Toc-siRNAadministration.

B: This figure shows that a 27/29-mer Toc-siRNA taken into the liver wascleaved by a dicer to produce a 21-mer siRNA.

FIG. 10 This figure shows that an efficiency of the apoB mRNA-expressionsuppression via a Toc-siRNA in a mouse liver is gene specific as well asdose dependent. Meanwhile, the data in FIG. 10 are all shown based onn=3, means plus/minus standard deviation (SE).

A: This figure shows that a Toc-siRNA targeted to the apoB gene reducesthe mRNA expression in the apoB gene-specific manner.

B: This figure shows the temporal change of the apoB-gene expression bya Toc-siRNA administration.

C: This figure shows that a Toc-siRNA has a dose-dependentexpression-suppressing activity.

FIG. 11 This figure shows that a 27/29-mer Toc-siRNA/LP taken into aHepa 1-6 cell line was cleaved by a dicer to produce a 21-mer siRNA.

FIG. 12 This figure shows that the apoB mRNA-expression-suppressioneffect via Toc-siRNA/LP in a mouse liver is gene specific as well asdose dependent. Meanwhile, the data in FIG. 12 are all shown based onn=3, means plus/minus standard deviation (SE).

A: This figure shows the reduction in the apoB mRNA expression in theliver due to a Toc-siRNA/LP administration.

B: This figure shows that a Toc-siRNA/LP targeted to the apoB genereduces the mRNA expression in the apoB-gene specific manner.

C: This figure shows that an apoB-siRNA/LP has a dose-dependentexpression-suppressing activity.

FIG. 13 This figure shows the effect of feeding prior to a Toc-siRNA/LPadministration. Meanwhile, the data in FIG. 13 are all shown based onn=3, means plus/minus standard deviation (SE).

FIG. 14 This figure shows the results of confocal microscopicobservations of liver segments after the rats were administered withfluorescently labeled Toc-siRNA/LP or Cho-siRNA/LP.

Left panel: This panel shows the result of using fluorescently labeledToc-siRNA.

Right panel: This panel shows the result of using fluorescently labeledCho-siRNA.

FIG. 15 This figure shows the results of a pathological analysis of theliver after a Toc-siRNA/LP administration.

FIG. 16 This figure shows the Toc-siRNA's expression-suppressingactivity for the liver apoB mRNA and the comparisons with other knownnon-viral vectors (however, the value for the HDL vector (Swiss) istaken from the data of the amount of the liver-apoB protein).

BEST MODE FOR CARRYING OUT THE INVENTION

The agent for suppressing a target gene expression of the presentinvention is characterized in that the agent contains nucleic acids forsuppressing a target gene expression, to which nucleic acids anintroduction substance into chylomicron is bound (hereinafter alsoreferred to as nucleic acids to which an introduction substance intochylomicron is bound), and that the agent is administered to avertebrate under a condition where the production of endogenouschylomicron is induced in the vertebrate.

Lipoproteins are categorized into chylomicron, VLDL, LDL, and HDLaccording to their specific gravities. Hepatic cells have receptors forthe above lipoproteins. These receptors are: an LRP-1 receptor thatbinds a chylomicron remnant which is a chylomicron metabolite; an LDLreceptor that binds LDL; and an SR-B1 receptor that binds HDL. Eachlipoprotein is taken into cells by receptor-mediated endocytosis.

Endogenous lipids are constantly taken in by LDL-LDL receptors andHDL-SR-B1 receptors, whereas exogenous lipids including vitamin Eabsorbed in large amounts in a short time postprandially are taken inmainly by chylomicrons, metabolized by LPLs (lipoprotein lipase) intochylomicron remnants, and subsequently taken in by hepatic cells viachylomicron-remnant receptors (LDL receptor-related protein 1; LRP-1receptor). An LRP-1 receptor on a hepatic cell moves to the cell surfacepostprandially to become activated.

This intake system into a hepatic cell that is mediated by a chylomicronremnant-LRP-1 receptor which is activated postprandially when absorbingan endogenous lipid, was used for the introduction of an siRNA.

When nucleic acids to which an introduction substance into chylomicronis bound is administered under a condition where the production ofendogenous chylomicron is induced after a lipid ingestion, theintroduction substance into chylomicron of the nucleic acids to which anintroduction substance into chylomicron is bound, interacts with achylomicron or the like, to form a complex of the nucleic acids to whichthe introduction substance into chylomicron is bound and thechylomicron. This complex of nucleic acids to which an introductionsubstance into chylomicron is bound and the chylomicron is taken into acell via an LRP-1 receptor, thereby the nucleic acids for suppressing atarget gene expression are taken into a cell in vivo, particularly intoa hepatic cell very sufficiently. Further, since a physiological systemis used, a significantly safer expression-suppressing agent can beobtained as compared to conventional suppressing agents. Meanwhile, FIG.1 shows the outline of the delivery of the present invention in the casewhere vitamin E is used as an introduction substance into chylomicronand an siRNA for suppressing the expression of the mouse apoB gene asnucleic acids for suppressing a target gene expression.

The above nucleic acids for suppressing a target gene expression can beany of natural nucleotides, modified natural nucleotides, or syntheticnucleotides as long as the nucleic acids have an activity of suppressinga target gene expression (hereinafter also referred to as “anexpression-suppressing activity”). The nucleic acids can be a DNA, RNA,or their chimeric forms, while by way of specific exemplifications, thenucleic acids are an siRNA, shRNA (short hairpin RNA), antisense,oligonucleotide, antagomir, nucleic acid aptamer, ribozyme, or decoy (adecoy molecule), among which an siRNA can be preferably exemplified. Byway of a specific exemplification, an siRNA that is targeted to themouse apoB gene is the siRNA consisting of the sense strand consistingof SEQ ID NO: 1 (GUCAUCACACUGAAUACCAAUGCUGGA) (27 mer) and the antisensestrand consisting of SEQ ID NO: 2 (UCCAGCAUUGGUAUUCAGUGUGAUGACAC) (29mer).

Further, it is preferable that the above nucleic acids are modified sothat they are not easily degraded in vivo. In particular, when thenucleic acids are an RNA, it is preferable that the nucleic acids havebeen subjected to an anti-RNase treatment such as a methylationtreatment or thiophosphorylation treatment so that the nucleic acids arenot easily degraded by RNases in a cell. More preferably, a nucleic-acidribose is methylated at its 2′ position or the skeletal binding of anucleic acid is thiophosphorylated. The number and the position ofnucleotide subjected to a methylation or thiophosphorylation mayslightly affect the expression-suppressing activity of the nucleic acidsand therefore, there is a preferred mode as to the number, position andthe like of nucleotide that is subjected to a methylation orthiophosphorylation. This preferred mode may vary depending on thenucleic-acid sequence that is to be modified and thus can not be statedcategorically, while such preferred mode can be found out easily byconfirming the expression-suppressing activity of the modified nucleicacids. For example, by way of exemplification, a preferred mode of ananti-RNase treatment of the siRNA consisting of the above-mentioned SEQID NO: 1 and 2 comprises: the methylations of the nucleotides ofnucleotide numbers 2, 5, 11, 15, 21, 24 and 25 of the sense strand (SEQID NO: 1) and the nucleotides of nucleotide numbers 1, 2, 5, 12, 14, 21,24, 25, and 26 of the antisense strand (SEQ ID NO: 2) at their 2′position of the ribose; the thiophosphorylation of the skeletal bindingof the nucleotide of nucleotide number 26 of the sense strand (SEQ IDNO: 1); and further methylations of the nucleotides of nucleotidenumbers 3, 4, 6, 27, and 28 of the antisense strand (SEQ ID NO: 2) attheir 2′ position of the ribose as well as the thiophosphorylation oftheir skeletal bindings.

The above phrase “an activity of suppressing a target gene expression”of nucleic acids means the activity that reduces the intracellularexpression of the target gene when the nucleic acids were introducedinto the cell as compared to the case without such an introduction. Thereduced intracellular expression of the target gene can be examined byquantifying the target gene mRNA or the protein encoded by the targetgene. By way of exemplification of the suppression level of the targetgene expression by the nucleic acids used in the present invention, whenthe nucleic acids are introduced into a given cell at 2 mg/kg, theintracellular expression of the target gene is 80% or less, morepreferably 60% or less, even more preferably 40% or less, and still morepreferably 20% or less at the mRNA level or at the protein level, ascompared to the case without such an introduction.

When the above nucleic acids are an siRNA, the number of nucleotides ofthe sense strand and/or the antisense strand may be 21, while it ispreferable that the number is more than 21, since an intracellular dicercleaves between the above introduction substance into chylomicron withapart of the siRNA and the siRNA (of 21 nucleotides), thereby the siRNAof 21 nucleotides can efficiently exercise its expression-suppressingeffect.

The above nucleic acids for suppressing a target gene expression can bedesigned by a known method based on the information on the target genesequence, or the part of the target gene sequence to which atranscription factor can bind. For example, nucleic acids forsuppressing a target gene expression can be designed using the methoddescribed in Japanese Laid-Open Patent Application No. 2005-168485 whenthe nucleic acids are an siRNA, the method described in Nature, 1990,346(6287):818-22 when the nucleic acids are a nucleic-acid aptamer, andthe methods described in FEBS Lett, 1988, 239, 285;Tanpakushitsu-kakusan-kouso (protein-nucleic acid enzyme), 1990, 35,2191; and Nucl Acids Res, 1989, 17, 7059 when the nucleic acids are aribozyme. Further, an antisense oligonucleotide, antagomir, or a decoycan be designed easily respectively based on the information on thetarget gene sequence, and the part of the target gene sequence to whicha transcription factor can bind.

The above nucleic acids can be prepared using a known method or thelike. For example, antisense oligonucleotide or a ribozyme can beprepared by determining the target sequence of an mRNA or earlytranscription product based on the cDNA sequence or genomic DNA sequenceof the target gene and synthesizing the sequence complementary to thetarget sequence using a commercially available DNA/RNA automaticsynthesizer (Applied Biosystems, Beckman and the like). Further, a decoyor siRNA can be prepared by synthesizing a sense strand and an antisensestrand respectively with a DNA/RNA automatic synthesizer, denaturing thestrands in an appropriate annealing buffer solution at about 90° C. toabout 95° C. for about 1 minute, and annealing at about 30° C. to about70° C. for about 1 to about 8 hours and the like. Further, anucleic-acid aptamer can be prepared by the method described in JapaneseLaid-Open Patent Application No. 2007-014292.

The above introduction substance into chylomicron is not particularlylimited as long as the substance can be taken into lipoproteins such asa chylomicron, and the preferred introduction substances are:fat-soluble substances such as carbon hydrides, higher alcohols, higheralcohol esters, higher fatty acids, higher fatty acid esters, sterols(in particular, cholesterols) and sterol esters; peptides such as cellpermeable peptides apolipoproteinsconjugate; and lipid-like moleculessuch as addition of alkylacrylatesor alkyl-acrylamides to primary orsecondary amines. Among these, a fat-soluble vitamin, which is anexogenous lipid that cannot be synthesized in vivo is more preferred,and vitamin E is particularly preferred because it is safer.

As the above vitamin E, tocopherols represented by the following generalformula (1) or tocotrienols represented by the following general formula(2), or mixtures containing two or more kinds of these compounds can bepreferably exemplified:

(wherein R¹ and R² represent a hydrogen atom or a methyl group, and R³represents a hydrogen atom or a carboxylic acid residue). Among these,particularly preferred are: an alpha-tocopherol (in general formula (1),R¹=methyl group, R²=methyl group, and R³=hydrogen atom); beta-tocopherol(in general formula (1), R¹=methyl group, R²=hydrogen atom, andR³=hydrogen atom); gamma-tocopherol (in general formula (1), R¹=hydrogenatom, R²=methyl group, and R³=hydrogen atom); sigma-tocopherol (ingeneral formula (1), R¹=hydrogen atom, R²=hydrogen atom, and R³=hydrogenatom); alpha-tocotrienol (in general formula (2), R¹=methyl group,R²=methyl group, and R³=hydrogen atom); beta-tocotrienol (in generalformula (2), R¹=methyl group, R²=hydrogen atom, and R³=hydrogen atom);gamma-tocotrienol (in general formula (2), R¹=hydrogen atom, R²=methylgroup, and R³=hydrogen atom); sigma-tocotrienol (in general formula (2),R¹=hydrogen atom, R²=hydrogen atom, and R³=hydrogen atom); and acetateesters and succinates of the above compounds. Among the above, analpha-tocopherol and gamma-tocopherol are particularly preferred.Further, anyone of d-, l-, and dl-form of vitamin Es may be usedlikewise.

Meanwhile, a combination of two or more kinds of the above introductionsubstances into chylomicron may be used. In addition, the introductionsubstance into chylomicron may be either a natural substance or asynthetic substance.

The bond between the above introduction substance into chylomicron andthe nucleic acids may be a direct bond or an indirect bond by anothersubstance mediating between them. Preferably, the bond is directlyformed by a chemical bond such as a covalent bond, ionic bond, orhydrogen bond, among which a covalent bond provides a more stable bondand therefore can be exemplified particularly preferably.

The method of binding the introduction substance into chylomicron andthe nucleic acids is not particularly limited. For example, when anintroduction substance into chylomicron and nucleic acids are covalentlybound, it is preferable that the covalent bond is formed according tothe method described in Tetrahedron Letters 33; 2729-2732, 1992, andwhen an ionic bond or a hydrogen bond is utilized, it is preferable toallow an arginine residue having a positive charge to bind to anintroduction substance into chylomicron and to utilize an ionic bond orhydrogen bond between this positive charge of the arginine residue and anegative charge of the nucleic acids such as an siRNA to form the bond.Meanwhile, from the viewpoint of obtaining a more stable binding to thenucleic acids, the number of arginine residues bound to an introductionsubstance into chylomicron is preferably at least two, more preferablyat least three, and even more preferably at least four.

In addition to the above essential components, theexpression-suppressing agent of the present invention can be formulatedwith components used in medicinal products as needed, such aslipoprotein, water, oil solution, wax, silicone, surfactant, alcohol,polyhydric alcohol, water-soluble high-molecular thickener, pH adjuster,flavor, antioxidant, chelating agent, pigment, antiseptic agent, andother medicinal components as well as inorganic or organic components,within the qualitative and quantitative range that does not affect theeffect of the present invention.

The expression-suppressing agent of the present invention can beformulated using the above essential components and optional componentsas needed according to a common method, into various dosage formsincluding solid formulations such as powder, granules, tablets, andcapsules; liquid formulations such as syrup, emulsion, and injections(including a subcutaneous injection, intravenous injection,intramuscular injection, and infusion); sustained-release formulationssuch as sublingual tablets, buccals, troches, and microcapsules;intraoral rapid-disintegrant; and suppository, among which injectionscan be preferably exemplified.

The expression-suppressing agent of the present invention isadministered to a vertebrate under a condition where the production ofendogenous chylomicron is induced in the vertebrate with a view toimproving the intake efficiency into a cell, of nucleic acids to whichan introduction substance into chylomicron is bound and thus increasingthe suppression efficiency of a target gene expression. The conditionwhere the production of endogenous chylomicron is induced in thevertebrate is not limited as long as the above object can be achieved,while a preferred condition is within 12 hours (for example, within 10hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours,and within 1 hour) after an oral lipid administration to a vertebrate.The lipid can be orally administered either as a lipid itself or in theform of a lipid-containing meal. It is preferable that the subject ofadministration is allowed to be in a state of starvation prior to aninduction of endogenous chylomicron. The detailed action mechanism hasnot been revealed how a lipid administration, and what is more, allowingthe subject of administration to be in a state of starvation, improvethe intake efficiency into a cell, of nucleic acids to which anintroduction substance into chylomicron is bound. However, consideringthe fact that an LRP-1 receptor on a hepatic cell, which is involved inlipoprotein intake, is known to be expressed at a higher level on cellmembranes and to be activated due to an oral lipid ingestion or byinsulin (Mol. Pharmacol. 2007 Jul. 3; 17609417), it is believed that alipid ingestion or the like causes the receptor involved in lipoproteinintake to be expressed at a higher level as well as activated, resultingin the increased chylomicron introduction into a hepatic cell andthereby improves the intake efficiency into a hepatic cell, of nucleicacids to which an introduction substance into chylomicron is bound.

Meanwhile, the above phrase “a state of starvation” refers to a state inwhich no food or drink (except a calorie-free drink or food such aswater) has been ingested for a certain period of time including, forexample, a state in which the subject of administration has not beengiven any food at least for 6 hours, preferably for at least 8 hours,more preferably at least for 12 hours, and even more preferably at leastfor 24 hours.

Sufficient LPLs are required in order for the administered chylomicronsto be metabolized into chylomicron remnants that will be rapidly takeninto hepatic cells. Therefore, it is preferable to administer LPLinhibitor such as Triton (for example, by an intravenous injection)before administering lipid (or a lipid-containing product) to increasethe concentration of chylomicrons. Further, LPLs act on hepatic cellsand causes them to take in vitamin E. With an administration of heparin,LPLs bound to heparan sulfate on the endothelial cell surface can befreed into the blood. Therefore, it is preferable to administer LPLand/or heparin to a vertebrate before nucleic acids are administered.

Further, the expression-suppressing agent of the present invention canbe administered by mixing ex vivo with a chylomicoron-rich lipoprotein.The lipoprotein is not particularly limited as long as the lipoproteincontains a lipid and apoprotein, and can be taken into a cell throughany lipoprotein receptors. A preferred lipoprotein contains cholesterol,triglyceride, phospholipid and apoprotein. The lipoprotein may becollected from a living body, a recombinant, or chemically synthesized,while a living body-derived lipoprotein is preferred from the viewpointof achieving a higher level of safety. Moreover, from the viewpoint offurther reducing side-effects due to an immunological allergic reaction,it is more preferable that the lipoprotein is collected from anindividual who is the subject of administration itself or from anindividual of the same species as the subject of administration of theexpression-suppressing agent.

By way of specific and preferred exemplification, the above livingbody-derived lipoprotein is: a chylomicron formed on the intestinalmoucosa by a lipid absorbed in vivo together with an apoprotein; or achylomicron remnant produced from the chylomicron degraded by vascularendothelial lipoprotein lipase. Among these, the chylomicron ispreferably exemplified. The above apoprotein is not particularly limitedand by way of preferred exemplification, the apoprotein is an apoBprotein or apoE protein.

A lipoprotein containing a chylomicron used in the present invention maybe collected from a living body, prepared from a synthetically-obtainedsubstance, or a mixture thereof. As for a method of collecting alipoprotein from a living body, the blood may be collected several hoursafter a lipid ingestion, while by way of preferred exemplification, 0.08to 0.4 g/kg of Triton is injected intravenously, subsequently 5 to 25ml/kg of high-protein/lipid solution (10 mg/ml of albumin, 40 mg/ml oftriolein, and 40 mg/ml sodium taurocholate) is administered orally to aliving body, and then blood is collected after 3 to 12 hours. Further,by way of preferred exemplification, the method of preparing the abovechylomicron-rich lipoprotein from a living body is, for example, amethod where serum collected from a living body several hours after alipid ingestion is added with a solution (a solution containing 11.4 gof NaCl, 0.1 g of EDTA, and 1 ml of 1N NaOH per 1 liter of water;specific gravity 1.006) in the same volume as the serum, and thesolution is centrifuged to provide a suspension from which thesupernatant is collected.

Further, the expression-suppressing agent of the present invention canbe administered orally or parenterally depending on the type of theabove-mentioned formulations, while from the viewpoint of achieving theexpression-suppressing effect more quickly and efficiently, theexpression-suppressing agent is preferably administered parenterally,more preferably administered by an injection, and even more preferablyadministered by an intravenous injection.

The applied dose of the expression-suppressing agent of the presentinvention varies depending on age, weight, symptom of the subject ofadministration, the kind of disease affecting the subject ofadministration, and the sequence of siRNA or the like contained in theexpression-suppressing agent. Generally, 0.1 to 30 mg/kg (siRNA weightbasis) can be administered by dividing the amount into 1 to 3 separatedoses a day.

The target disease of the expression-suppressing agent of the presentinvention is not particularly limited as long as the suppression of aparticular gene could lead to the treatment of the disease. The abovedisease is caused by the expression of a particular pathologic targetgene, and by way of particularly preferred exemplification, the diseaseis viral hepatitis caused by an elevated expression of hepatitis virusgene or the familial amyloid neuropathy caused by the expression of atransthyretin gene variant.

The subject of administration in the present invention is notparticularly limited as long as the subject is an animal. By way ofpreferred exemplification, the subject of administration is vertebrates,while animals belonging to mammals or birds are more preferablyexemplified, among which, humans, rats, mice, pigs, rabbits, dogs, cats,monkeys, horses, cows, goats, and sheep can be even more preferablyexemplified and human is particularly preferably exemplified.

Further, the expression-suppressing agent of the present invention canbe used as an active ingredient of the pharmaceutical composition of thepresent invention. The pharmaceutical composition of the presentinvention is characterized in that it contains theexpression-suppressing agent of the present invention as an activeingredient.

In the present invention, the method for delivering nucleic acids into acell (hereinafter also referred to as the delivery method of the presentinvention) is characterized in that the method comprises process (C) ofadministering to a vertebrate nucleic acids for suppressing a targetgene expression, to which nucleic acids an introduction substance intochylomicron is bound, under a condition where the production ofendogenous chylomicron is induced in the vertebrate. When nucleic acidsto which an introduction substance into chylomicron are bound areadministered to a vertebrate under a condition where the production ofendogenous chylomicron is induced in the vertebrate, the introductionsubstance into chylomicron of the nucleic acids to which an introductionsubstance into chylomicron is bound, interacts with a lipoprotein toform a complex of the nucleic acids to which an introduction substanceinto chylomicron is bound and the lipoprotein. This complex of thenucleic acids to which an introduction substance into chylomicron isbound and the lipoprotein, is taken into a cell via a lipoproteinreceptor, and therefore has enabled a very efficient in vivointracellular delivery of nucleic acids for suppressing a target geneexpression. The use of the delivery method of the present invention hasalso enabled nucleic acids to be delivered appreciably more safely ascompared to conventional methods even in vivo. The terms “nucleic acidsfor suppressing a target gene expression, to which nucleic acids anintroduction substance into chylomicron is bound” and “a lipoprotein” inthe delivery method of the present invention have meanings as describedabove.

In the delivery method of the present invention, the method ofadministering to a vertebrate nucleic acids to which an introductionsubstance into chylomicron is bound, under a condition where theproduction of endogenous chylomicron is induced in the vertebrate, isnot particularly limited, and the nucleic acids can be administered bythe same method as the expression-suppressing agent of the presentinvention.

Tissues that can be delivered nucleic acids to which an introductionsubstance into chylomicron is bound by the delivery method of thepresent invention is not particularly limited, and the nucleic acids towhich an introduction substance into chylomicron is bound can bedelivered in vivo to any tissues such as liver, brain, peripheralnerves, lungs, intestinal tract, pancreas, kidneys, cardiac muscles, andskeletal muscles.

FIG. 2 shows a diagram of an alpha-tocopherol transportation in the bodyto show that nucleic acids to which a lipid-soluble substance is boundcan be delivered in vivo to each tissue by the delivery method of thepresent invention.

As shown in FIG. 2, an alpha-tocopherol contained in food or drink isabsorbed by small intestines and further, mainly taken into achylomicron which is a kind of lipoprotein. This chylomicron ismetabolized into a chylomicron remnant by lipoprotein lipase (LPL) andis transported to the liver by this chylomicron remnant. In the hepaticcytoplasm, an alpha-tocopherol is taken into a VLDL (very low-densitylipoprotein) by an alpha-TTP (alpha-Tocopherol Transfer Protein).Subsequently, the VLDL containing an alpha-tocopherol is secreted intothe blood and metabolized into an LDL (low-density lipoprotein) or HDL(high-density lipoprotein cholesterol) to form analpha-tocopherol-containing LDL or HDL. These LDL and HDL aretransported by blood to each tissue so that the alpha-tocopherol isefficiently taken into various tissues through a receptor-mediatedendocytosis that is mediated by a lipoprotein receptor existing on eachtissue (for example, an LRP-1 receptor, LDL receptor, HDL receptor orthe like). Incidentally, even when a substance other thanalpha-tocopherol is used as an introduction substance into chylomicronin the present invention, nucleic acids to which an introductionsubstance into chylomicron is bound of the present invention togetherwith a lipoprotein are efficiently taken into a cell of each tissuethrough an endocytosis mediated by a lipoprotein receptor.

The delivery method of the present invention is not particularly limitedas long as the method comprises the above process (C), while from theviewpoint of enhancing the delivery efficiency of the nucleic acids soas to achieve an increased suppression efficiency of a target-geneexpression, it is preferable that, before an administration of thenucleic acids to a vertebrate under a condition where the production ofendogenous chylomicron is induced, the method comprises administeringheparin and/or LPL to a subject of administration (process (B)); andthat, before process (C), the method further comprises allowing avertebrate to be in a state of starvation (process (A)). The above term“a state of starvation” refers to a state where no food or drink isingested for a certain period of time (except calorie-free food or drinksuch as water). The above state of starvation comprises the state whereno food is given to the subject of administration, for example, at leastfor 6 hours, preferably at least for 8 hours, more preferably at leastfor 12 hours, and even more preferably at least for 24 hours.

The method for treating a disease of the present invention (hereinafteralso referred to as the treatment method of the present invention) ischaracterized in that the method comprises process (C) of administeringto a vertebrate nucleic acids for suppressing a target gene expression,to which nucleic acids an introduction substance into chylomicron inbound, under the condition where the production of the endogenouschylomicron is induced in the vertebrate. When nucleic acids to which anintroduction substance into chylomicron is bound are administered to avertebrate under a condition where the production of endogenouschylomicron is induced in the vertebrate, as mentioned above, thenucleic acids to which a lipid-soluble substance is bound are taken intoa cell in vivo very efficiently, thereby an effect of suppressing thetarget gene expression is sufficiently exhibited in an appreciably safermanner as compared to conventional methods. Consequently, a superiortherapeutic effect is obtained against the disease. In the treatmentmethod of the present invention, the phrases “nucleic acids forsuppressing a target gene expression, to which nucleic acids anintroduction substance into chylomicron is bound” and “achylomicron-rich lipoprotein” have meanings as described above.

In the treatment method of the present invention, the method ofadministering to a vertebrate nucleic acids to which an introductionsubstance into chylomicron is bound, under a condition where theproduction of endogenous chylomicron is induced in the vertebrate is notparticularly limited, and the nucleic acids can be administered by thesame method as the expression-suppressing agent of the presentinvention. Further, the target disease of the treatment method of thepresent invention is not particularly limited as long as the disease iscaused by an elevated expression of a given gene. By way of specificexemplification, the disease is same as the above target diseases of theexpression suppressing agent of the present invention.

The present invention will be described in detail with reference to thefollowing examples, while the scope of the present invention will not belimited to these exemplifications.

EXAMPLE Example 1 Cell Culture

A mouse hepatic-cancer cell line (Hepa 1-6 cell line) used in theexperiment described hereinbelow was maintained in the following manner.

A mouse hepatic-cancer cell line (Hepa 1-6 cell line) was maintainedunder the condition of 37° C. and 5%-by-mass of CO₂, using a growthmedium (DMEM: Sigma) added with 10%-by-mass of bovine fetal serum, 100units/ml of penicillin and 100 micro-g of streptomycin.

Example 2 Separation of Lipoprotein-Rich Serum with High ChylomicronContent

The lipoprotein-rich serum with high chylomicron content used in theexperiment described hereinbelow was separated and adjusted in thefollowing manner.

12- to 14-week old ICR mice (Charles River Laboratories: U.S.A) wereintravenously injected at the tail with 0.4 g/kg of Triton WR-1339(Nacalai Tesque, Inc.: Kyoto, Japan). 10 minutes after the injection,the above mice were orally administered with 0.5 ml of protein-richfluid food containing 5 mg of bovine serum albumin (Sigma), 20 mg oftriolein (Wako Pure Chemical Industries, Ltd.: Tokyo, Japan), and 20 mgof sodium taurocholate (Wako Pure Chemical Industries, Ltd.: Tokyo,Japan). To the serum collected from the mice 3-12 hours after the oraladministration of the protein-rich fluid food, a solution (a solutioncontaining 11.4 g of NaCl, 0.1 g of EDTA, 1 ml of 1N NaOH per 1 liter ofwater; specific gravity 1.006) was added in the same volume as theserum, which was then centrifuged at 26,000 g for 30 minutes under thecondition of 16° C. The upper one-sixth of the resultant suspension wascollected as a lipoprotein-rich serum. Triglyceride in thislipoprotein-rich serum was measured using Triglyceride E-TST kit (WakoPure Chemical Industries, Ltd.: Tokyo, Japan), and the amount used wasadjusted as desired.

Example 3 Isolation of the Liver of the Mice Administered with Toc-siRNAand the Like

The liver of the mice administered with Toc-siRNA and the like used inthe experiment described hereinbelow was prepared in the followingmanner.

First, 4-week old ICR mice (Charles River Laboratories: U.S.A) wereprovided. The above mice were fasted for 24 hours prior to theadministration of Toc-siRNA and the like. Then, the mice wereintravenously injected at the tail with 0.08 to 0.2 g/kg of Triton.Subsequently, the above mice were orally administered with 0.5 ml ofprotein-rich fluid food containing 250 micro-g of bovine serum albumin(Sigma) and 1 mg of triolein (Wako Pure Chemical Industries, Ltd.:Tokyo, Japan). Ten minutes to 10 hours from the administration of theprotein-rich fluid food, the mice were given at the tail a singleintravenous administration of 0.25 ml of a 10%-by-mass maltose solutioncontaining Toc-siRNA or the like. Ten minutes before administration ofToc-siRNA, the mice were intravenously injected at the tail with 8 to 10units of heparin. Then, the mice were anesthetized as desired by anintraperitoneal administration of 60 mg/kg of pentobarbital, killed byperfusing a PBS solution transcardially, and the liver was taken out.

Example 4 Quantitative RT-PCR

Unless otherwise described, the quantitative RT-PCR performed in theexperiment described hereinbelow was performed in the following manner.

The total RNA was extracted from the cultured cells or tissues of themice using Isogen (Nippon Gene Co., Ltd.: Tokyo). By using SuperscriptIII and Random hexamers (Invitrogen) as directed by the accompanyingprotocol, the above RNA was reversely transcripted to obtain the DNAwhich is complementary to the RNA. A quantitative RT-PCR was performedusing 0.5-micro-1 of the above-mentioned complementary DNA, apredetermined primer and TaqMan Universal PCR Master Mix (AppliedBiosystems) as directed by the accompanying protocol. In the abovequantitative RT-PCR, the amplification was performed using ABI PRISM7700 Sequence Detector (Applied Biosystems) by repeating 40 cycles eachconsisting of a denaturalization at 95° C. for 15 seconds and anannealing at 60° C. for 60 seconds. Meanwhile, the primer for the mouseapoB gene, the primer for the GAPDH gene, and the primer for the TTR(Transthyretin) gene used as the above-mentioned predetermined primerswere designed by Applied Biosystems.

Example 5 Northern Blot Analysis

Unless otherwise described, the northern blot analysis performed in theexperiment described hereinbelow was performed in the following manner.

Using MirVana (Ambion, Inc.: Austin, Tex., U.S.A), the total RNA wasextracted from the Hepa 1-6 cell line or the mouse liver. The extractedRNA was concentrated using Ethachinmete (Nippon Gene Co., Ltd.: Tokyo,Japan). From the obtained RNA, 2 micro-g was electrophoreticallyseparated with 14%-by-mass polyacrylamide gel (containing urea) and thentransferred onto a Hybond-N+ membrane (Amersham Biosciences, Inc.:Piscataway, N.J., U.S.A).

Meanwhile, as a probe for the northern blotting, an siRNA antisensestrand, fluorescently labeled with Gene Images 3′-oligo labeling kit(Amersham Biosciences, Inc.), was provided.

Using the above-mentioned transfer membrane and the probe and the like,a northern blot analysis was performed in accordance with a commonmethod. The signals from the above fluorescent labels were visualizedusing Gene Images CDP-star detection Kit (Amersham Biosciences, Inc.)

Example 6 siRNA Synthesis

Based on the mouse apoB gene sequence, an siRNA targeted to the mouseapoB gene was designed. The nucleotide sequence of the sense strand (27mer) of this siRNA is shown in SEQ ID NO: 1(GUCAUCACACUGAAUACCAAUGCUGGA) and the nucleotide sequence of theantisense strand (29 mer) in SEQ ID NO:2(UCCAGCAUUGGUAUUCAGUGUGAUGACAC). These nucleotide sequences of the sensestrand and the antisense strand were synthesized in accordance with acommon method.

Subsequently, these nucleotide sequences were modified for an improvedstability to in vivo RNases. More specifically, the nucleotides ofnucleotide numbers 2, 5, 11, 15, 21, 24, and 25 of the siRNA sensestrand (SEQ ID NO: 1) and the nucleotides of nucleotide numbers of 1, 2,5, 12, 14, 21, 24, 25, and 26 of the antisense strand (SEQ ID NO: 2)were subjected to 2′-O-ribose methylation; the skeletal bond of thenucleotide of nucleotide number 26 of the sense strand (SEQ ID NO: 1)was subjected to thiophosphorylation; and further, the nucleotides ofnucleotide numbers 3, 4, 6, 27, and 28 of the antisense strand (SEQ IDNO: 2) were subjected to 2′-ribose methylation as well asthiophosphorylation of their skeletal bond.

Subsequently, in accordance with the method described in a literature(Tetrahedron Letters 33; 2729-2732. 1992), an alpha-tocopherol (TokyoChemical Industry Co., Ltd.: Tokyo, Japan) was bound to the 5′ terminalof the siRNA antisense strand (FIG. 4).

The above antisense-strand nucleotide sequence to which analpha-tocopherol is bound and the sense-strand nucleotide sequence wereannealed in an RNase-free distilled water (DW) at 95° C. for 1 minuteand then incubated at 37° C. for 1 hour, thereby thealpha-tocopherol-bound siRNA (hereinafter also referred to as“Toc-siRNA”) used in the present study was obtained. Meanwhile, thissiRNA has the sense-strand nucleotide sequence and the antisense-strandnucleotide sequence that are different in length as described above, andthis has created a 2-mer-nucleotide overhung at the 3′ terminal of theantisense strand (FIG. 3A).

Further, according to a common method, cholesterol-bound siRNA(hereinafter, also referred to as “Cho-siRNA”) wherein cholesterol isbound to 5′ terminal of the antisense strand of the above siRNA wasprepared using 3′-cholesteryl-TEG-CPG (GlenResearch), which is acholesterol-modification reagent for RNA.

Example 7 siRNA Stability Test

To examine the effect of the above chemical modification in Example 6 onthe siRNA stability, the following experiment was performed.

First, an siRNA targeted to the mouse apoB gene and chemically modifiedin the above Example 6 (hereinafter also referred to as “a modifiedsiRNA”) was prepared (2 micro-g). This siRNA was incubated at 37° C. for24 hours in distilled water (DW) or in the above lipoprotein-rich serumprepared in accordance with the method described in Example 2. A givenquantity was taken from each of the obtained solutions, treated withprotainase K for 1 hour, and subjected to an electorophoresis with2%-by-mass agarose gel, respectively.

Further, in place of the above modified siRNA, an siRNA same as theabove siRNA except that it had not been modified (hereinafter alsoreferred to as “an unmodified siRNA”) (2 micro-g) was used to perform anelectrophoresis in the same manner.

The result of the electrophoresis is shown in FIG. 3B. As can be seenfrom FIG. 3B, the modified siRNA shows a significantly improvedstability in the serum as compared to the unmodified siRNA (NakedsiRNA). This demonstrates that the modified siRNA is more stable toRNAases contained in the serum as compared to the unmodified siRNA.

Example 8 In Vitro Activity Test of siRNA

A chemical modification of an siRNA sometimes impairs theexpression-suppressing activity (silencing activity) of the siRNA. Toexamine the in vitro expression-suppressing activity of the abovemodified siRNA, the following experiment was performed.

First, the above modified siRNA (2 micro-g) targeted to the mouse apoBgene was prepared. Next, a Hepa 1-6 cell line was transfected with 10 nMof the above modified siRNA using Lipofect Amine RNAiMAX (Invitrogen).The transfected cell line was cultured for 24 hours after thetransfection. From the resultant cell line, the total RNA was extractedand measured for the amount of endogenous apoB mRNA by the abovequantitative RT-PCR described in Example 4.

Further, by using an unmodified siRNA or an siRNA to a non-targeted gene(control siRNA) in place of the above modified siRNA, a quantitativeRT-PCR was performed in the same manner.

The results are shown in FIG. 3C. As can be seen from FIG. 3C, themodified siRNA exhibited an expression-suppressing activity which iscomparable to that of the unmodified siRNA (Naked siRNA).

Example 9 Interaction of Toc-siRNA and Lipoprotein

To examine whether the Toc-siRNA is taken into a lipoprotein (LP)-richserum with high chylomicron content, the following experiment wasperformed.

First, 45 micro-1 of aqueous solution of 0.4 micro-g/ml Toc-siRNA wasincubated for 1 hour together with 135 micro-1 of lipoprotein-rich serumcontaining 2.7 mg of triglyceride prepared in accordance with the abovemethod described in Example 2 to prepare an alpha-tocopherol-boundsiRNA-containing lipoprotein (Toc-siRNA/LP) solution. This Toc-siRNA/LPsolution was filtered with a centrifugal filtration unit, Micron YM-100(Millipore) that blocks a material having the molecular weight of100,000 or above. The solution obtained by the filtration was subjectedto an electrophoresis with 2%-by-mass agarose gel and the RNA was dyedwith ethidium bromide.

Further, in place of the above “Toc-siRNA/LP solution”, itsalpha-tocopherol bond-lacking form of “siRNA/LP solution”, itslipoprotein-lacking form of “Toc-siRNA solution”, and itsalpha-tocopherol bond-lacking and lipoprotein-lacking form of “siRNAsolution” were used to go through the same operation.

Further, for each of the above cases, a solution obtained by eluting thefraction left on the filter after the filtration was used in place ofthe solution obtained by the filtration, to go through the sameoperation.

The results of these experiments are shown in FIG. 5. The upper row inFIG. 5 shows the results of an electrophoresis of a solution obtained byeluting the fraction left on the filter (in centrifugal device) and thelower row shows the results of an electrtophoresis of a solutionobtained by filtration (Filtered un-conjugated siRNA). As can be seenfrom the results shown in FIG. 5, siRNA is trapped by the above filteronly in the case where the siRNA has been bound to an alpha-tocopherol(Tocopherol-conjugation+) and incubated with a lipoprotein-rich serum(lipoproteins+). This shows that the interaction of an siRNA and alipoprotein is created only when the siRNA has been bound to analpha-tocopherol (Tocopherol-conjugation+) and incubated with alipoprotein-rich serum (lipoproteins+), and thus suggests thepossibility that a Toc-siRNA is taken into a chylomicron and the like invivo and transferred to and absorbed by each tissue via receptors suchas an LRP-1 receptor.

Example 10 Interaction of Toc-siRNA or Cho-siRNA and a Lipoprotein

(1) Gel Electrophoresis Experiment

Rat chylomicron collected from a lymph vessel of a rat administered withhigh-fat food was adjusted with PBS so that the triglycerideconcentration becomes 40 mg/ml and then treated with 100 μg/mllipoprotein lipase (LPL) to obtain an aqueous solution of ratchylomicron remnant (hereinafter, referred to as CR). 10 μl of the CRaqueous solution was mixed with 2 μl of a 50 μM Toc-siRNA aqueoussolution and then the mixed solution was electrophoresed with 15%acrylamide gel. The result is shown in the second lane from the left ofFIG. 6. Further, 10 μl of the above CR aqueous solution was mixed with 2μl of a 50 μM Cho-siRNA aqueous solution and then electrophoresed in asimilar manner. The result is shown in the far right lane of FIG. 6.Further, as a control, the result of similarly electrophoresed siRNAalone is shown in the far left lane and the second lane from the right.

As is clear from FIG. 6, in all cases of siRNA alone, it is migratedtowards the downstream, while the mixtures with CR became unmigrated inboth cases of tocopherol-bound and cholesterol-bound siRNAs. This isconsidered to be resulted from the binding of siRNA and CR which has alarge diameter and has no electric charge and therefore resulted in nomigration.

(2) Fluorescence Correlation Spectroscopic Experiment

Rat chylomicron prepared in the similar manner as in the above Example10 (1) was mixed with fluorescently labeled cholesterol-bound siRNA ortocopherol-bound siRNA and then the diffusion time was measured byfluorescence correlation spectroscopy (FCS) with reference to aliterature (Biochemistry 41; 697-705. 2002). The measurement results areshown in FIG. 7.

As is clear from FIG. 7, the diffusion time became nearly 30 timeslonger in the case of a complex added with chylomicron as compared tothe case of siRNA alone. Since the diffusion time is proportional to thediameter of a measured substance, it is considered that the diameter ofthe substance comprising siRNA has become larger due to the binding withchylomicron. Supposing that the length of siRNA is in the vicinity of 6nm, the diameter of the complex of a group added with chylomicron isapproximately 180 nm (theoretical value). The diameter of chylomicronitself (FIG. 8) measured by dynamic light scattering (DLS) was almostconsistent with this value.

Example 11 In Vivo siRNA Delivery by Toc-siRNA

To examine whether the Toc-siRNA conveys its siRNA in vivo, thefollowing experiment was performed.

First, in accordance with the above method described in Example 3, aToc-siRNA labeled with Cy3 fluorescence dye was administered to a mouse.After a lapse of 1 hour, the mouse was killed and the liver was takenout.

Next, a part of the liver was fixed in a 4%-by-mass paraformaldehyde/PBSsolution for 6 hours, and immersed in a 30% sucrose/PBS solutionovernight at 4° C. The fixed liver was embedded in OCT compound (SakuraFinetek Japan Co., Ltd.: Tokyo, Japan), frozen by liquid nitrogen, andthen segmented to 4 micro-m thickness with Leica CM3050 Cryostat (Leica:Germany). The frozen segment was moved onto Super Frost plus Microscopeglass slide (Fisher Scientific: Pittsburgh, Pa., U.S.A), counterstainedfor 20 minutes using 13 nM Alexa-488 Phalloidin (Invitrogen)/PBSsolution and 40 nM Topro-3 (Invitrogen)/PBS solution. Then the segmentwas enclosed in a vector shield (Vector: Burlingame, Calif., U.S.A) andobserved with LSM 510 confocal scanning microscopy (Zeiss: Germany)(FIG. 9A).

FIG. 9A is an observation of the neighboring area of the liver sinusoid.The Cy3 fluorescence-dye signals were generally detected intensely onthe part surrounding the blood vessel (the right part of the imagedivided by the dashed line in FIG. 9A), and it was confirmed that siRNAswere introduced into the hepatic cells (by way of example, the cellsshown by triangles in FIG. 9A) and nonparenchymal cells (by way ofexample, the cells shown by arrows in FIG. 9A). This demonstrates that aToc-siRNA is capable of conveying its siRNA to the liver.

Example 12 Efficient Toc-siRNA Processing In Vivo

To examine whether a Toc-siRNA taken into a hepatic cell is processedinto a matured form of siRNA, the following experiment was performed.

First, in the same manner as in the above method described in Example 3,the liver of a mouse 1 hour after a Toc-siRNA administration wasprovided, and the detection of siRNA was performed in accordance withthe above method described in Example 5 (FIG. 9B). As can be seen fromthe result of FIG. 9B, a processed 21-mer siRNA was confirmed to existin addition to the original 27/29-mer siRNA. This shows that theToc-siRNA was taken into a hepatic cell and then cleaved from the27/29-mer siRNA into the 21-mer siRNA by a dicer existing in thecytoplasm.

Example 13 Animal Testing Related to Gene-Silencing by Toc-siRNA

To examine the ability of a Toc-siRNA to reduce a target gene expressionin vivo, the following experiment was performed.

First, in the same manner as in the above method described in Example 3,the mouse was administered with the Toc-siRNA, killed after a lapse of24 hours, and the liver was taken out. Next, in accordance with theabove-method described in Example 4, a quantitative RT-PCR wasperformed, and mRNA expressions were measured for each of the apoB gene,GAPDH gene, and TTR gene. The results are shown in FIG. 10A. As can beseen from FIG. 10A, in the case of a Toc-siRNA using an siRNA targetedto the mouse apoB gene (apoB-1 Toc-siRNA), the apoB mRNA expression wassignificantly reduced (n=3, P<0.001) as compared to the case of aToc-siRNA using an siRNA unrelated to the apoB (control Toc-siRNA) orthe case of a solvent only (maltose). Further, the relative mRNAexpressions (relative expression based on the total RNA) of otherendogenous genes (GAPDH gene, TTR gene) expressed on the liver were inthe same range as compared to the case of administering the controlToc-siRNA or maltose only. The above fact demonstrates that theToc-siRNA (apoB-1 Toc-siRNA) specifically reduces only the target geneexpression in the liver and that it does not affect non-specific geneexpressions, i.e., that the Toc-siRNA does not have an off-targeteffect.

Next, the timings of the samplings from the Toc-siRNA-administered micewere staggered and the time-dependent change was measured. The resultsare shown in FIG. 10B. As can be seen from FIG. 10B, the continuity ofthe apoB-gene expression-suppressing activity by the apoB-1 Toc-siRNAwas observed until 2 days after the Toc-siRNA administration, while byday 4, the expression returned to the same level as others (controlToc-siRNA, maltose).

Further, a similar experiment was performed by gradually changing thedose of Toc-siRNA. The results are shown in FIG. 10C. The apoB-1Toc-siRNA's expression-suppressing activity for the apoB gene was dosedependent. The dose of 32.0 mg/kg resulted in an activity of at least80% expression suppression.

Example 14 Confirmation Test for Toc-siRNA-Related Side Effects

In accordance with the above method described in Example 3, theToc-siRNA-administered mice were examined for side effects.

Specifically, blood was collected from the mice 3 days after theadministration of 2 mg/kg Toc-siRNA/LP, measured for the white bloodcell count (WBC), blood platelet count (Plt) in the blood and wasbiochemically analyzed on the total amount of protein (TP),aminotransaminase (AST, ALT) and blood urea nitrogen level (BUN).

Further, the interferon (IFN) induction was examined in the mice 3 hoursafter the administration of 2 mg/kg Toc-siRNA/LP. First, an ELISA kitwith the detection limit of 12.5 pg/ml (PBL Biochemical Laboratories,Biosource) was used to measure the interferon-alpha (IFN alpha)concentration in the serum, but no IFN alpha was detected. Additionally,an attempt was made with an RT-PCR to confirm the expression ofinterferon-beta in the liver of the mice administered with 2 mg/kg ofToc-siRNA, but none was detected.

The results of the above experiments are shown in Table 1. The abovefacts show that the Toc-siRNA causes almost no side effects, and thatthe gene suppression by the Toc-siRNA is not caused by an interferonresponse.

IFN-α, BUN, TP, AST, ALT, WBC and Plt levels in mouse serum afterintravenous injection of Toc-siRNA or maltose. * IFN-α BUN TP AST ALTWBC Plt Treatment (pg/ml) (mg/dl) (g/dl) (U/l) (U/l) (/μl) (×10⁴/μl)apoB1  3 h <12.5 Toc-siRNA 24 h 19.1 ± 1.0 5.1 ± 0.1 78 ± 1 21 ± 3 2800± 330 122.0 ± 0.3 48 h 24.0 ± 2.4 5.5 ± 0.1 67 ± 4 22 ± 1 2600 ± 550112.2 ± 18.9 maltose  3 h <12.5 24 h 22.0 ± 0.9 5.5 ± 0.1 79 ± 9 25 ± 22600 ± 560 117.9 ± 13.8 48 h 24.5 ± 1.5 5.5 ± 0.1 60 ± 3 26 ± 3 3700 ±900 109.0 ± 7.0 * Values represent mean ± S.E. (n = 3)

Example 15 Preparation of an Alpha Tocopherol-Bound-siRNA- orCholesterol-Bound-siRNA-Containing Lipoprotein

A lipoprotein-rich serum with high chylomicron content provided inaccordance with the above method described in Example 2 was adjustedwith 10%-by-mass maltose solution to obtain a triglyceride concentrationof 20 g/L. Toc-siRNA (1 micro-g/micro-1, in terms of the amount ofsiRNA) was mixed with the same volume of the lipoprotein-rich serum (20g/L triglyceride concentration). The mixture was incubated at 37° C. for1 hour, and an alpha-tocopherol-bound-siRNA-containing lipoprotein wasobtained (hereinafter also referred to as “Toc-siRNA/LP”). Further,cholesterol-bound-siRNA-containing lipoprotein (hereinafter, alsoreferred to as “Cho-siRNA/LP”) was obtained in the similar manner exceptfor using Cho-siRNA in place of Toc-siRNA.

Example 16 Efficient Toc-siRNA/LP Processing In Vitro

To examine whether a Toc-siRNA/LP taken into a cell is processed into amatured form of siRNA, the following experiment was performed.

First, a Hepa 1-6 cell line having been cultured in a medium without atransfection reagent was added with 100 nM Toc-siRNA/LP and then furthercultured for 6 hours.

Using this Hepa 1-6 cell line, a northern blot analysis was performed inaccordance with the above method described in Example 5. The result isshown in FIG. 11. As can be seen from FIG. 11, a processed 21-mer siRNAwas confirmed to exist in addition to the original 27/29-mer siRNA. Thisshows that a Toc-siRNA/LP can be taken into a cell of the Hepa 1-6 cellline and that the 27/29-mer siRNA was cleaved into a 21-mer siRNA by adicer existing in the cytoplasm.

Example 17 Animal Testing Related to siRNA Delivery and Gene-Silencingby Toc-siRNA/LP

To examine the Toc-siRNA/LP's ability to deliver its siRNA in vivo orthe ability to reduce a target gene expression in vivo, the followingexperiment was performed.

First, in accordance with the above method described in Example 3, amouse was administered with a Toc-siRNA/LP and killed after a lapse of 2days and the liver was taken out. Using this liver, a quantitativeRT-PCR was performed in accordance with the above method described inExample 4, and mRNA expressions were measured for each of the apoB gene,GAPDH gene, and TTR gene. The results are shown in FIG. 12. As can beseen from FIG. 12A, in the case of a Toc-siRNA/LP using an siRNAtargeted to the mouse apoB gene (ApoB Toc-siRNA/LP vector), the apoBmRNA expression was significantly reduced (n=3, P<0.001) as compared tothe case of a Toc-siRNA/LP using an siRNA unrelated to the apoB(Unrelated siRNA/LP) or the case of LP only (control (LP only)).Further, the results of FIG. 12B show that this expression-reductioneffect is apoB-gene specific. More specifically, even when aToc-siRNA/LP using an siRNA targeted to the apoB gene was administered,the relative mRNA expressions (relative expression based on the totalRNA) of other endogenous genes (GAPDH gene, TTR gene) expressed on theliver were in the same range as compared to the case of administering LPonly.

Subsequently, the same experiment was performed by gradually changingthe dose of Toc-siRNA/LP. The results are shown in FIG. 12C. TheToc-siRNA/LP's expression-suppressing activity (knockdown effect) forthe apoB gene was dose dependent. Even the dose of 1.0 mg/kg resulted inan activity of at least 80% expression suppression.

Further, to examine the effect of feeding after fasting (administrationof protein-rich fluid food) on the expression-suppressing effect ofToc-siRNA/LP, an expression-suppressing activity was measured in a casewhere the protein-rich fluid food was not administered in the aboveexperiment. The results are shown in FIG. 13. As can be seen from FIG.13, the expression-suppressing activity was reduced by 31% in the casewithout feeding (Feeding (−)) as compared to the case with feeding(Feeding (+)). This suggests that a Toc-siRNA/LP is taken in via anLRP-1 receptor expressed on the liver, which receptor is activated byfeeding to take in chylomicron remnants.

Example 18 SiRNA Delivery with Toc-siRNA/LP or Cho-siRNA/LP byIntraperitoneal Administration to Rat

Fluorescently-labeled Toc-siRNA/LP or Cho-siRNA/LP was prepared inaccordance with the description of the above Example 15 by labelingsiRNAs with Cy3 fluorescent dye, which was then administeredintraperitoneally at 8 mg/kg in terms of the nucleic acid amount to 3week-old SD rats (Charles River Japan: Kanagawa, Japan). 8 hours after,the liver segments were observed under a confocal microscope, and theresults are shown in FIG. 14. The fluorescent dye was found to exist inalmost all hepatic cells in both cases of Toc-siRNA/LP or Cho-siRNA/LPadministration. No significant difference was observed between the twocases.

Example 19 Confirmation Test for Toc-siRNA/LP-Related Side Effects

As with the above Example 14, a biochemical analysis and the like wereperformed also on the blood of a mouse administered with Toc-siRNA/LP(Table 2).

In addition, the liver was taken out from the Toc-siRNA/LP-administeredmouse in the above method described in Example 3. A part of the liverwas fixed in 4%-by-mass paraformaldehyde, embedded in paraffin, and a 4micro-m-thick segment was produced. The segment was subjected to ahematoxylin-eosin staining for a pathologic analysis. Further, as acontrol, results of a hematoxylin-eosin staining are shown for the liversegment of a mouse administered with LP only in place of Toc-siRNA/LP(FIG. 15).

As stated above, no particular abnormality was observed in both thebiochemical analysis of the blood and pathological analysis of the livertissues of the Toc-siRNA/LP-administered mouse.

Further, as with the above Example 14, the interferon induction wasexamined in the Toc-siRNA/LP-administered mouse, but none was detected.

The above facts show that, as with the above Toc-siRNA, a Toc-siRNA/LPcauses almost no side effects.

TABLE 2 [Biochemical analysis and cell count of the blood] Biochemicalanalysis and cell count of the blood TP Alb T-Bil BUN Cre Na K (g/dl)(g/dl) (mg/dl) (mg/dl) (mg/dl) (mEq/l) (mEq/l) Toc- 5.3 ± 0.2 3.4 ± 0.20.05 ± 0.01 28.4 ± 1.5 0.14 ± 0.02 151 ± 4 5.5 ± 0.3 siRNA/LP LP only5.1 ± 0.1 3.4 ± 0.1 0.05 ± 0.01 22.7 ± 2.3 0.13 ± 0.01 151 ± 1 4.7 ± 0.4AST ALT LDH Alp RBC WBC Plt (U/l) (U/l) (U/l) (U/l) (×10⁴/μl) (/μl)(×10⁴/μl) Toc- 57 ± 5 21 ± 1 278 ± 6 541 ± 58 791 ± 22 6300 ± 600 122 ±3 siRNA/LP LP only 48 ± 2 17 ± 1 258 ± 27 467 ± 53 816 ± 13 6600 ± 200 92 ± 13

Example 20 Comparisons with Other Non-Viral Vectors

In vivo efficiency of apoB mRNA-expression suppression in a mouse liverwas compared between other already reported non-viral vectors (Nature432:173-178, 2004; Nature 441; 111-114, 2006; ACS Chem Biol 2; 237-241,2007; Proc Natl Acad Sci USA 104; 12982-12987, 2007, NatureBiotechnology 25:1149-1157, 2007). The results are shown in FIG. 16. ThesiRNAs used in other known viral vectors are same as the siRNA used inthe Toc-siRNA or Toc-siRNA/LP of the present invention, and are targetedto the same region of the mouse apoB mRNA (Nature 432:173-178, 2004).Further, as the data for the Toc-siRNA of the present invention, thevalue of apoB-1 Toc-siRNA (1 day after the administration of 2.0 mg/kg)in the above FIG. 10B of Example 13 are used, and as the data for theToc-siRNA/LP of the present invention, the value of Toc-si RNA/LP (2days after the administration of 1.0 mg/kg) in the above FIG. 12C ofExample 17 were used.

The results of FIG. 16 show that the present invention, in particularthe Toc-siRNA/LP, is highly superior as compared to other siRNA deliverysystems, in terms of the required amount of siRNA and itsexpression-suppressing activity.

INDUSTRIAL APPLICABILITY

According to the method of delivering nucleic acids of the presentinvention, nucleic acids such as an siRNA for suppressing a target geneexpression can be delivered in vivo more safely and efficiently.According to the expression-suppressing agent and pharmaceuticalcomposition utilizing the delivery method of the present invention, theexpression of a specific gene that causes a disease can be suppressed invivo more safely and efficiently. Further, according to the treatmentmethod of the present invention utilizing the delivery method of thepresent invention, the expression of a specific gene that causes adisease can be suppressed in vivo more safely and efficiently andconsequently, the disease can be treated more safely and efficiently.

1. A complex comprising: a. nucleic acids for suppressing a target geneexpression, wherein the nucleic acids are hound to vitamin E; and b. achylomicron.
 2. The complex according claim 1, wherein the nucleic acidsare one or more kinds of nucleic acids selected from the groupconsisting of siRNA, shRNA, antisense oligonucleotide, antagomir,nucleic-acid aptarner, ribozyme, and decoy.
 3. The complex according toclaim 2, wherein the nucleic acids are siRNA.
 4. The complex accordingto claim 3, wherein the nucleic acids are RNA subjected to an anti-RNasetreatment.
 5. The complex according to claim 4, wherein the anti-RNasetreatment is 2′-O-methylation treatment and/or thiophosphorylationtreatment.
 6. A method for in vivo delivering nucleic acids forsuppressing a target gene expression, comprising the following step (C):(C) binding vitamin E and the nucleic acids and administering the sameto a vertebrate, wherein a production of endogenous chylomicron orchylomicron remnant is induced in the vertebrate.
 7. The methodaccording to claim 6, wherein the step (C) comprises binding vitamin Eand the nucleic, acids and administering the same to a vertebrate within12 hours after the vertebrate is administered with a lipid.
 8. Themethod according to claim 7, wherein the lipid is orally administered.9. The method according to claim 6, further comprising the followingstep (A′) before step (C): (A′) administering triton to a vertebratebefore a production of endogenous chylomicron or chylomicron remnant isinduced in the vertebrate.
 10. The method according to claim 6, furthercomprising the following step (B) before step (C): (B) administering LPLand/or heparin to a vertebrate before administering the nucleic acids tothe vertebrate.
 11. The method according to claim 6, further comprising,the following step (A) before step (C): (A) allowing a vertebrate to bein a state of starvation prior to the administration of a lipid.
 12. Themethod according to claim 6, wherein step (C) comprises administering toa vertebrate the nucleic acids bound to vitamin E after mixing the samewith concentrated chylomicron obtained from the vertebrate.
 13. Themethod according to claim 12, further comprising the following step (B)before step (C): (B) administering LPL, and/or heparin to a vertebratebefore administering the nucleic acids to the vertebrate.
 14. The methodaccording to any one of claims 6 to 13, wherein the nucleic acids areone or more kinds of nucleic acids selected from the group consisting ofsiRNA, shRNA, antisense oligonucleotide, antagomir, nucleic-acidaptamer, ribozyme, and decoy.
 15. The method according to claim 14,wherein the nucleic acids are siRNA.
 16. The method according to claim15, wherein the nucleic acids are RNA subjected to an anti-RNasetreatment.